This project is developed in response to the Program Announcement PA-07-362 [Exploratory Cancer Prevention Studies Involving Molecular Targets for Bioactive Food Components (R21)] to identify novel bioactive compounds in pomegranate juice that may act synergistically with the ellagitannins. There is growing evidence to suggest that approximately one third of all cancer deaths can be prevented by changes in diet and lifestyle and/or through the use of preventive drug therapy. Identification of novel agents capable of modulating molecules responsible for cancer cell survival and metastasis is warranted. Available methodologies limit the detection to known compounds with potential chemopreventive properties. Unrecognized minor components that may be more potent remain untested which limits the exploitation of the power of chemoprevention. Pomegranate, which comprises of ellagitannins, including punicalagins and ellagic acids, has gained attention due to its superior antioxidant properties. The metabolites of ellagitannins accumulate the most in prostate tissue when administered orally and bioavailability studies have focused on measuring ellagic acid in plasma. However, in vitro studies have shown that pomegranate juice has lower IC50 values than individual components. Minor components of pomegranate include phenolic acids, flavanoids and tannins besides others, known widely for their protective effects. We hypothesize that the minor components of pomegranate juice act synergistically with the ellagitannins and is more efficacious in modulating molecules involved in preventing prostate cancer. Support to our hypothesis lies in the fact that ellagic acid when tested in a mammary tumor model was not as efficacious as black raspberry powder, which contains 150-fold less ellagic acid. Further analysis of pomegranate extract detected several components apart from the major ellagitannins, which were not reported previously. We used hybrid mass spectrometer (MS-filtered Fourier- transform Ion Cyclotron Resonance MS) with a nanospray autosampler that has the advantage of high mass measurement accuracy wit high resolving poser and a large dynamic range. The other advantage of this instrument is the identification of compounds in mixtures with ultra-high resolution and accuracy, without need for chromatographic separation. Data mining identified several polyphenols not reported so far in pomegranate juice which are shown to have antiviral and anti-inflammatory properties, among others. We will therefore pursue the following specific aims: 1) Identification of minor polyphenols, bioavailability and toxicity of major and minor polyphenols from pomegranate extract using FTICR-MS;and 2) Efficacy of various fractions of pomegranate extract in modulating the tumor growth in a xenograft model. These studies will identify the components of juice other than the well studied ellagitannins and determine the synergistic effects. Based on the information on bioavailability and efficacy, future studies will focus on isolation of minor components and identification of molecular targets and mechanisms. PUBLIC HEALTH RELEVANCE: Pomegranate contains multiple compounds with varied biological activities. Most of the studies focus on major components such as ellagitannins, while the total juice seems more protective. This project will identify the minor bioactive components in pomegranate that might work synergistically to elicit their efficacy. The data derived from this project will form the basis to understand the mechanisms of action of pomegranate juice components in prostate cancer prevention.